Identification

and characterization of the gene for neurofibromatosis type 1 Ray White

Howard

Hughes

Medical

Institute

and Peter O’Connell

and University

of Utah School

Elucidation of the partial genomic structure gene responsible for neurofibromatosis type its function, have led to new opportunities this particular disease process, but also for involved in cellular growth and Current

Opinion

in Genetics

of Medicine,

Salt Lake City, Utah,

USA

and DNA sequence of the 1, and discovery of clues to not only for understanding clarifying signalling pathways differentiation.

and Development

1991, 1:15-19

the disease process, all recommended the NF~ gene as a for the mapping approach to its identification.

Introduction

candidate

As predicted by Knudson [ 11, certain genes responsible for inherited cancers play an important role in noninherited forms of the same diseases, through somatic mutation. Furthermore, even after inheritance of a recessive mutation in a given cancer gene, somatic mutation of the normal homologue is often an important step in carcinogenesis [2]. In recent years, genetic mapping approaches have provided initial chromosomal localizations that have led to the identiIication and cloning of new cancer genes [ 3,4 1. Mapping by genetic linkage in family studies depends upon the availability of DNA-based genetic markers. Genetic markers are tested in order to lind one with a segregation pattern that matches that of the inherited cancer gene, thereby providing an approximate chromosomal location [5]. More precise mapping through the use of additional closely spaced markers, often taking advantage of chromosomal rearrangements, can result in the deflnition of a relatively short genomic segment. DNA probes from this small segment are used to identify transcribed genes that can then be scanned for cancer-specific mutations. Neurofibromatosis type 1 (NFl), the von Recklinghausen or peripheral form of neurolibromatosis, was an early target for the mapping approach just described. NFl is characterized primarily by tumors in the peripheral nervous system and by cafe-au-lait spots on the skin, but the clinical manifestations are variable. Individuals with NFl carry a signilicamly Increased risk of malignancies, especially in nerve tissue. Interestingly, in as many as half the cases, the mutation has occurred as a de novo event in a germline cell; this high mutation rate is only one of many puzzling aspects of NFl [6]. The high rate of incidence of NFl (about 1 in 3500 among all ethnic groups), its highly penetrant, autosomal dominant pattern of inheritance, and the lack of any biochemical knowledge about

CAP-GTPase-activating Current

The search for the A/f7 gene

The first report of genetic linkage of the NFI gene with a known DNA segment placed it near the centromere of human chromosome 17 [7]. These initial linkage results were quickly confirmed [8], and localized the gene to within a region composed of some tens of millions of bps of DNA, and thus large enough to contain hundreds of genes. Further studies localized the NFI gene more precisely to the proximal long arm of chromosome 17 [9,101. A direct approach to identification of the gene was enabled by the discovery of two NFl patients, each carrying a translocation of chromosome 17: t(1;17) in one case [ll] and t(I7;22) in the other [12]. In both cases, the chromosome 17 breakpoint was in the same region previously identified by the linked markers. In order to map the translocation breakpoints efficiently, rodent x human hybrid cell lines were established in which the derivative translocation chromosome 17’s were the only human DNA in a rodent background [12,13]. Because *it seemed likily that each of the mutations caused by the translocations was responsible for the disease by interrupting the NFI gene, the gene was expected to span both of the breakpoints. Genomic DNA clones encompassing the translocation breakpoints should, therefore, contain exons of the NFZ gene, and could be used to pinpoint a relatively small region, from which DNA probes useful in identifying candidate transcripts could be developed. To expedite the process of isolating DNA clones from the vicinity of the NFl translocation breakpoints, a panel of microcell-mediated, chromosome-transfer somatic cell hybrids that contained subfragments of chromosome 17

Abbreviations protein; NFl-neurofibromatosis Biology

Ltd ISSN 0959-437X

type

1.

15

16

Genetics

of disease

was constructed [141,Characterization of these ceII lines using tightly linked genetic markers identied those with a chromosome 17 subfragment that ought to include the NFl gene. DNA probes developed from these hybrids permitted the construction of a Iine structure map of the region [9], and identified large restriction fragments spanning the translocation breakpoints [15,161. A chance finding provided the most significant meansof accessto the iVFI region, however. A mouse leukemia gene, .&i-2, had been mapped to murine chromosome 11 [ 171; the homologues of genes carried on this chromosomeare found on chromosome 17 in humans.Mapping studiesdemonstrated that the human homologue of Evi-2 is located between the two NFl translocations.USing the EL72 locus as a starting point, overlapping fragments of genomic DNA in the breakpoint region were isolated as cosmid clones and arranged into a physical map. This map indicated that the translocation breakpoints are some 50 000bp apart and spanned by the cosmid contig [ 18*]. Very importantly, these cloned genomic segmentsprovided the meansto screen cDNA libraries for clones derived from altered NH genes.Although the first found in the region, now called EVQA, was an obvious candidate NFl gene, no deletions or other changeswere found initiaIiy to distinguish the version present in NFl patients from that found in una.Ifectedindividuals [19]. Two further genes were then discovered within the breakpoint region, EV12B and OMGP [20,21]. OMGP seemed an especially promising candidate NFl gene becauseit encodesoligodendrocyte-myeIin glycoprotein, an important component of the myelin sheath that is elaborated by the oligodendrocytes of the central nervous system [221. Like ElLG-4, however, neither gene could be shown to be mutated in NFl patients, even at the level of single bp changes. Further examination of DNA from additional NFl patients led to the detection of three NFl -specificdeletions, present within the region disrupted by the two known translocations. The smallest of these deletions had removed an 11kb segment of genomic DNA without disrupting any of the known genes, but including a region with strong homology to murine DNA This sequence conservation signaledthe presenceof yet another candidate gene; in fact, part of the conserved region was also present in a 3.8 kb genomic fragment of known sequence previously found to span the t(17;22) breakpoint. Using this fragment to probe a murine macrophage cDNA library, a single clone was isolated which, in turn, was used to identify numerous positive clones from a human fetai brain cDNA library The cDNA contig defined by theseclones crossesthe t(17;22) translocation breakpoint and hasbeen partially deleted by the 11 kb deletion described above [23**]. Because the corresponding genomic sequence was aiready known, the boundariesof severalexons within the cDNA sequencewere readily deiined [240*]. Severalsingle bp mutations, expected to render the predicted peptide non-functional, were found in the coding regions of

cDNA inserts derived from NFl patients. These iindings, together with the previous evidence that the sequenceof the new transcript spanned the t(17;22) breakpoint and the inference that the encoded transcript in aii probability alsoextended past the t(1;17) breakpoint, led to the conclusion that the NFl gene had been identiIied [23**,24°0]. Independent support for this interpretation was provided the concomitant description of two cDNA clones hybridizing to the 3.8 kb EcoRI fragment that contains the t( 17;22) breakpoint [ 25*=]. These cDNA clones had been identified using a ‘jump’ clone derived from one of the EK’ZA cosmids [18-l. In addition, a new mutation consistingof a 500 bp insertion, present in an NFl patient but not in either of his parents, was also found in the EcoRI fragment. Although it was ultimately shown that the large transcript dehned by these cDNA clones encompassed neither the t( 17;22) breakpoint nor the site of the 500 bp insertion [ 261, the sequencedata indicated that the transcript was indeed identical to the one we had reported [23**,24**]. Thus, the 500bp insertion mutation must Lie within the NFI gene. Additional support for the conclusion that the newly identified transcript was encoded by the NFl gene came from the observation that hybrid cells carrying one or other of the two translocation chromosomesfailed to produce the large transcript. Subsequent experiments have shown that the insertion mutation is an A/u1 repeat and is located in an intron of the NFl gene; its functional effect is to alter the splicing pattern in such a way that an exon is dropped from the processed transcripts (M Wallace, personal communication). It is now apparent that the NFl gene is relatively large, with a transcript size estimated at 11-13 kb, the numerous exons being scattered over at least 350 kb of genomic DNA A single, large intron of the NFl gene encompassesall three of the genes previously localized between the translocation breakpoints, EWA, Ek72B and OIMGP. Interestingly, the NFl gene is transcribed in the opposite orientation with respect to these three interdigitated genes, i.e. towards the telomere. This phenomenon has no precedent in human genetic literature other than one report of a single gene of unknown function embedded within an intron of the Factor VIII gene, which has additional copies elsewhere in the genome [27]. Interdigitated genes have been reported more frequently in Drosophila. For example, the dunce locus (involved in conditioned responsememory) contains at least two developmentally regulated transcriptional units, Sgs1 and pig 1 [28]. Interestingly, the Gall locus of D. mehnogaster (involved in purine biosynthesis) contains a developmentally regulated pupai cuticle protein gene transcribed in the opposite direction [29]. This organization is conserved in the distantly related D. pseudo obscuru, and some evidence exists to indicate coordinated expression of these two genes,despite their totally different functions [30]. The normal control of gene expression for the single-copy interdigitated genes at the NFI locus remainsto be investigated.Moreover, how the expression of thesethree genesmight be affected by mutations in NFl, which could account for variability in the clinical manifestations of the disease,is a question for further research.

Identification

Characteristics

and characterization

of the gene for neurofibromatosis

of the NFl gene

AI-I initial search of protein sequence databases for similarities to the peptide predicted from the first 4000 bp of the NFl transcript sequence revealed no strong similarities to known proteins. An additional 3000 bp of sequence lying 5’ to this initial segment, however, revealed an intriguing similarity in amino acid sequence between the predicted NFZ gene product and the catalytic domains of the mammalian GTPase-activating proteins (GAPS) and their yeast counterparts, IRA1 and IRA2 [31**]. Furthermore, examination of the amino acid sequences outside the catalytic domains of the IRA1 and IRA.2 genes indicated that the ammo acid similarities extend for some 300 ammo acids in the N-terminal direction, and for more than 800 amino acids in the C-terminal direction. The implications of these similarities are profound, as this family of genes is known to be involved in the mechanisms that control cell growth and differentiation through their interaction with the ras gene family. GAP activity substantially accelerates the hydrolysis of Ras-bound GTP to GDP, thereby converting the Ras protein from the active to the inactive form. When NFl is altered by mutation, the normal interaction of its protein product with the rasgene product, for example, may be disrupted; such a mechanism might well contribute to tumorigenesis.

type 1 White and O’Connell

Similarity in amino acid sequence, however, is not rigorous proof of functional homology, biochemical and genetic experiments are needed to establish the relationship. Fortunately for the hypothesis, the catalytic domain of the NFl gene product is indeed capable of interacting with both the mammalian rasand the yeast Mgene products. The NFI peptide segment, consisting of the 350 amino acids 4th sequence similarity to the GAP catalytic domain, accelerates decay of GTP bound to Ras protein in vitro as well as complementing I.1 and I. mutants in yeast [32**,33**,34**]. The functional homologies between the NFZ peptide and the GAP and IRA proteins suggest two general possibilities for the molecular pathophysiology of NFl. As seen in Fig. l(a), the normally functioning NFl gene product may downregulate a Ras-mediated growth signal; akematively, as shown in Fig. l(b), the product of the normal NFZ gene could serve as an effector of a Ras-mediated differentiation signal. In either case, loss of the NFl product would result in abnormal cell growth. Either hypothesis, by analogy with the retinoblastoma gene, could be consistent with enhancement of the mutant phenotype through loss of the normal NFZ allele. With the model that the major role of the NFZ gene is to downregulate a growth signal, the presence of the nonmutant allele might provide almost normal levels of gene

23 * Unregulated growth signal

FiR. 1. Two models for the oathoohvsiology of a defect in NH. (a) NFI as a negative regulator of the growth-stimulating pathway. Loss of functional NFl peptide would permit unregulated growth fright). fb) NFl as an effector in the differentiation pathway. Loss of functional NFI peptide would inhibit cell differentiation fright).

17

18

Genetics

of disease

activity and, therefore, near normal regulation of Ras activity. LOSS or mutation of the non-mutant allele would then be necessary for the development of a more extreme phenotype. With the model that the major role of the NF1 gene is to propagate a differentiation sig nal, the presence of the non-mutant allele might provide adequate gene activity to propagate the signal. If this model is correct, emergence of the mutant phenotype in a cell may also require loss of the normal allele. The evidence concerning chromosomal loss associated with tumors in NFl patients seems somewhat inconsistent to date, although remaining suggestive. The most common NFI tumor, the neurofibroma, does not readily permit a direct test of the chromosome loss hypothesis, because it contains a mixture of cell types, the identity of the cell type bearing the primaty responsibility for tumor development being uncertain. Furthermore, any such studies are complicated by the fact that there is a well documented propensity for the loss of the region of chromosome 17 associated with the p53 gene, located on 17~. In contrast, several of the malignancies associated with neurofibromatosis, such as neurofibrosarcomas, astrocytomas, and pheochromocytomas, do afford an opportunity to examine the chromosome loss hypothesis.. In neurofibrosarcomas, complete loss of chromosome 17 or partial loss of 17~ only has been observed in some studies [35], whereas others have found evidence for the specific loss of chromosome 17q at a significant frequency 1361. A recent study of adrenal tumors demonstrated the specific loss of 17q among some NFl patients, but failed to document losses in pheochromocytomas obtained from non-NFl patients [ 371. Concluding

cessive

References and recommended Papers of special interest, published have been highlighted: . of interest .. of outstanding interest

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by Chromosomal

3.

remarks

The discovery and characterization of the NFl gene offers the promiseof new meansto diagnoseand study this disease.New researchapproachesmay ultimately lead to an effective means of medical intervention. As is often the case in the study of rare human genetic disorders, however, the final significancemay be even broader becausethe gene product hasnow been specifically placed within the category of proteins that act in cells to control growth and differentiation. The NFl protein is, therefore, of great interest to investigatorsworldwide who are seeking to understand normal cellular mechanismsaswell as the altered signal transduction pathways that lead to the formation of tumors.

Alleles

toma.Nature1983,305779-784.

DC, of

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Map-

Identification

and characterization

of the gene for neurofibromatosis

Two Translocation Breakpoints Associated with von Recklinghausen Neurofibromatosis. Genomics 1990, 7547-554. Overlapping cosmkls derived From human homologue of a murine oncogene were used to ‘walk’ through the region between two NFl breakpoints. Cosrnid walking permitted definition of the breakpoint region to within 50-60 kb.

28.

CHEN

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Oligodendrocyte-

Belongs fo a Distinct Family of Prothe HNK-l Carbohydrate. / Cell Biol

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Deletions and a Translocation Interrupt a Cloned Gene at the Neurofibromatosis m 1 Locus. CeN 1990, 62:187-192. New mutations were detected in three NFl patients within the translocation~breakpoint region. The smallest of the three deletions disrupted none of the other genes in the region, but did include part of a novel cDNA later shown to span one of the NFI breakpoints. This is strong evidence that this new cDNA clone represents part of the NFl gene.

24. ..

CAW~HON R, WEISS R, Xu G, VISKOCHII. D, CULVER M, STEVENS J, ROBERTSON M, DUNN GES~F~AND R, O*CONNEU P, WHITE R:

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M, &IARCHUK A, Fouwm

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26.

WALIACE

M, MARCHUK

rofibromatosis 27.

LEV~NZON

Gene:

B, KENWRICK

..

D, ANDERSEN

I+ COLLINS

Correction.

Scieme

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M, FECHTEL

Within a Proteins

K, FRI~TROM J: Gene

HENIKOFF

S, ECHTEDAIZADEH

M: Conserved

at the Drosophila

Xu G, O’CONNEIL CULVER M, DUNN

Cart

Locus.

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D,

GESTEIAND

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Domain of tbe 1 Gene Product Stimulants rus GTira Mutants of S. cereuisiae. Ceil

Neurofibromatosis Type Pase and Complements 1990, 63~835-841. Expression of the NFl domain of homology with GAPS suppressed the heat-shock.sensitive phenotype of yeast iral and iru2 mutants. The NFl domain stimulated the GTPase activity of yeast Ra52 and human H-5 proteins. 33. ..

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D, GCIUAG G, MCCABE L, &IRK R, O’CONNELL

P. CRAZIER P, CAWIHON

W, R,

m AL: The GAP-Related Domain of the Neurofibromatosis ljpe 1 Gene Product Interacts with ras ~21. Cell 1990, 63:843-849. Expressing a fragment of rhe GAP-related NFl gene product stimulated the GTPase activity of wild-type ~21~ protein but not of its oncogenic murants. The affinity of the NFl product for p21m was about 20.fold higher than that of GAP, but its specific activity was lower. 34. ..

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BAIJ&TER R, WIGLER

SAUMO Encodes

4

LETCHER a Rotein

Functionally Related to Mammalian GAP and Yeast IRA Proteins. Cell 1990, 63:851-859. Biological assays showed interaction of the NFl gene product with Ras proteins and demonstrated structural and functional similarities and differences among the GAP, and IRAl, IR42, and NFl-encoded proteins. 35.

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R, ODEH H, J, MITCHEU

A, CAL: Type 1 Neurofibromatosis Gene: Identication of a Large Transcript Disrupted in Three NFI Patients. Science 1990, 249181-186. A transcript from the NFl breakpoint region identified by ‘chromosome jumping’; a patient with NFl wds found to have a NFl-specific 500 bp insertion near the gene encoding this nanscript. The tnnscdpi is apparently also interrupted by two NFI-specific translocations.

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.. J, STEFANS~~N

tvpe 1 White

of the

G JR, GIT~CHIER

R White,

Human

ment of Human lake City, Utah

Factor

Howard

Hughes

Medical

Genetics, University 84132, USA

Institute, of Utah

and P O’Connell, School

of Medicine,

DepartSalt

Identification and characterization of the gene for neurofibromatosis type 1.

Elucidation of the partial genomic structure and DNA sequence of the gene responsible for neurofibromatosis type 1, and discovery of clues to its func...
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